The secreted protein, Sonic hedgehog (Shh), plays an integral role in establishing the ventral midline of the mouse central nervous system (CNS) through inductive signaling from the underlying axial mesoderm. In the absence of Shh function, ventral midline development is perturbed resulting in holoprosencephaly, a structural malformation of the brain, as well as neuronal patterning and path finding defects along the length of the anteroposterior neuraxis. Central to the understanding of floor plate development are the mechanisms by which Shh transcription is established in the ventral midline of the mouse CNS. As an entry point towards identifying the genes directly regulating Shh expression, we systematically screened 1 Mb of DNA surrounding the Shh locus for its ability to target reporter gene expression to sites of Shh transcription in the ventral midline of the mouse neural tube. This analysis uncovered five enhancers distributed over 400kb whose combined activity covered all sites of Shh transcription in the mouse embryonic CNS from the ventral forebrain to the posterior extent of the spinal cord. Experiments described in this proposal will address specific questions related to the regulation and function of individual Shh enhancers. We will explore the independent mechanisms by which two functionally redundant Shh floor plate enhancers are controlled. Candidate transcriptional regulators for both enhancers have been identified in screens for DNA binding proteins. The requirement of these candidate transcription factors in the regulation of their respective enhancers will be investigated by genetic experiments in the mouse as well as by biochemical experiments using embryonic stem cell derived floor plate cultures. Additional experiments relating to the targeted inactivation of selected enhancers in mice, will not only reveal their requirement in regulating Shh transcription but will also provide a unique opportunity to determine the relative contribution of Shh signaling from selective sites within the CNS, something that hasn't been readily possible in Shh-/- embryos given the complexity of their CNS phenotype. Results from these experiments are predicted to have important implications for the mechanisms underlying floor plate induction as well as providing insight into novel causes of holoprosencephaly.